Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote underreplication is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling underreplication and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote underreplication. Our work provides new mechanistic insight into the process of underreplication and its links to RT. Overall design: Replication timing profiles prepared in salivary gland from female 3rd instar larvae prior to wandering and in fat body from female larvae 96 hours after egg laying (AEL).

DNA复制与拷贝数的调控对于维持基因组稳定性、保障细胞与组织功能不可或缺。DNA复制受时序调控，这一过程被称为复制时序（Replication Timing, RT）。Rif1是RT的关键调控因子，在多倍体细胞的拷贝数调控中发挥核心功能。在既往研究（Munden等，2018）中，我们证实Rif1可与SUUR协同，抑制复制叉推进并促进特定基因组区域的复制不足（underreplication）。但Rif1依赖的RT调控如何参与其介导的复制不足过程，目前尚不明确。本研究通过在果蝇多倍体细胞中采用计算方法测定RT，发现SUUR与Rif1在调控复制不足与RT方面具有差异化功能。研究结果显示，Rif1可通过在SUUR的上下游同时发挥作用，进而促进复制不足。本研究为复制不足过程及其与RT的关联提供了全新的机制解析。
实验整体设计：分别从徘徊期前的雌性三龄幼虫唾液腺，以及产卵后96小时（After Egg Laying, AEL）的雌性幼虫脂肪体中制备复制时序分析图谱。